The botulinum neurotoxins (BoNTs), produced by Clostridium botulinum are among the most potent toxins known to man. The Centers for Disease Control and Prevention (CDC) has classified it as a potential bioweapon, Category A, because of its extreme potency and lethality, its ease of production and transport, and the need for prolonged intensive care among affected persons. Recently, it has been observed that the majority of therapeutic and diagnostic BoNT programs are creating narrow spectrum reagents (molecules specific for only one very specific subtype of BoNT) and that the number of subtypes is much larger than originally anticipated. This project focuses on determining the three-dimensional structure of prioritized serotypes and subtypes of BoNTs. Serotype/subtype information is critical in the development of broad spectrum therapeutics and diagnostics that are needed. The overall goal is structurally characterizing these variants at the protein level and developing effective countermeasures through the creation of new antibody reagents and small molecule inhibitors via collaborations. This study will conduct a series of X-ray crystallographic studies to determine the three dimensional structure of the prioritized holotoxin serotypes/subtypes, prioritized catalytic and binding domain serotype/subrypes, and complexes with antibodies and small molecule inhibitors. In addition to the structures, the project will also produce highly purified protein samples that will be made available to its collaborators and other workers in the biodefense consortium for assay and antibody development.